Purification And Immunogenicity Of Norovirus GⅡ.17 Virus Like Particles

Objective:Norovirus GⅡ.17 virus like particles were expressed in Kluyveromyces marxianus and purified.The immunogenicity of norovirus GⅡ.17virus like particles under different inoculation routes and mucosal adjuvants was investigated by immunizing BALB/c mice.Methods:（1）Kluyveromyces marxianus expression system was screened for high expression strains of norovirus GⅡ.17 virus like particles by SDS-PAGE and Western blot;（2）Norovirus GⅡ.17 virus like particles were purified using a monomix core 1000 composite chromatography column in tandem with a Q column,and the purification efficacy was examined and the morphology of virus like particles was observed by SDS-PAGE followed by transmission electron microscopy;（3）BALB/c females mice between aged 6-8 weeks were selected and divided into 11groups and each group is 5,and the purified norovirus GⅡ.17 virus like particles were sorted into 20μg each mouse received three doses at weeks 0,2,and 4.Groups with different inoculation route were set up as intranasally/intranasally/intranasally,subcutaneous/subcutaneous/subcutaneous,intramuscular/intramuscular/intramuscular,intranasally/subcutaneous/subcutaneous,and intranasally/intramuscular/intramuscular（Al（OH）₃adjuvant for all groups）;The different immune adjuvant groups were set up as Al（OH）₃,Cp G ODN 1826,poly I:C,Al（OH）₃+Cp G ODN 1826,and Al（OH）₃+poly I:C groups（all groups received the nose dropping route）;（4）The serum of mice at 0 weeks,2 weeks,4 weeks,6 weeks and 8 weeks and the alveolar lavage fluid of mice at 8 weeks were collected for Ig G/Ig A antibody level and Ig G antibody titer determination.Spleens and lymph nodes were harvested from mice at week 8,and flow cytometry was performed for T lymphocytes,B lymphocytes,and the cytokine IFN-γ,TNF-αand IL-4 was analyzed.Results:（1）Obtained a high expression strain of norovirus GⅡ.17 virus like particles;（2）Norovirus GⅡ.17 virus like particles were successfully purified using a Monomix core 1000 composite chromatography column coupled with a Q column,and the yield could reach 0.57 g/L;（3）Purified norovirus GⅡ.17 virus like particles could effectively induce immune responses in BALB/c mice.Ig G antibody levels of different immunization routes:with the increase of immunization times and time,the antibody levels of the other groups except for the nose dropping route gradually increased,and all of them reached and maintained at a high level at weeks 6-8;The intramuscular and subcutaneous routes were used to provoke a significant antibody response at 2 weeks,and the intramuscular route had higher antibody levels than the subcutaneous route.The level of antibody by intramuscular route alone reached the highest level at week 6,which was higher than the rest of the groups,but it decreased at week 8 compared with week 6,suggesting that intramuscular route could induce rapid and high level of immune response in mice.Antibody titers of different immunization routes:the highest antibody titer at week 6 of intramuscular route alone was higher than that of the remaining groups,and the highest titer reached 819200;The first dose of nasal drip,intramuscular route of booster at week 8 was the highest,and the highest titer reached 3276800,suggesting that the first dose of nasal drip,intramuscular route of booster may induce higher levels of immune response,but it takes longer.Levels of Ig G antibody with different adjuvants:the adjuvant alone group increased gradually with the time between the time of immunization and the time of immunization,in which the Cp G adjuvant group was higher than the rest of the groups at both the 6th and 8th weeks.The antibody levels decreased from week 6to week 4 in the group with combined adjuvants,and both persisted at low levels for weeks 2-8 in the Al（OH）₃adjuvanted group,even lower than that in the group without adjuvants,suggesting that Al（OH）₃cannot enhance the immunity of norovirus GⅡ.17VLPs and even inhibit this immunity.Titers of antibodies with different adjuvants compatibility:at 6th week,Cp G adjuvant group showed the highest titer,which reached 409600;At week 8,the Al（OH）₃+Cp G adjuvanted group had the highest antibody titer,which reached 1638400;It suggests that Cp G adjuvant could enhance the mice immunity to norovirus GⅡ.17 VLPs.Cytokine levels:the number of CD3-/CD19+cells was highest in the spleen of mice immunized by the intramuscular route.In part,it can induce IFN-γand TNF-αproduction by T cells in the spleen and lymph nodes of mice.These results suggested that intramuscular route could enhance the Th1 cell response to some extent.IL-4 was more produced by T cells in the spleen of mice by the subcutaneous route of immunization,and the proportion of IL-4-secreting T cells in the lymph nodes was the highest in the first dose nasal drip and subcutaneous booster immunization groups,suggesting that the subcutaneous route may enhance Th2 cell responses in mice.The highest number of CD3-/CD19+cells was observed in the spleen of mice treated with Cp G adjuvant,which also secreted IFN-γof T cells accounted for the highest percentage,whereas the Al（OH）₃+Cp G group mice secreted TNF-αin the spleen of T cells had the highest proportion,suggesting that Cp G could enhance the level of cellular immune response to norovirus GⅡ.17 VLPs in mice to some extent.Conclusion（s）:A high expression system for GⅡ.17 norovirus VLPs was constructed in Kluyveromyces marxianus;The use of intramuscular injection with GⅡ.17 norovirus VLPs allows rapid acquisition of potent protective antibodies;Cp G adjuvant could effectively enhance the cellular immune responses of VLPs of GⅡ.17norovirus VLPs.This study provides a reference for the expression,inoculation route,and mucosal adjuvants of Norovirus VLP.